Imbalance or defects in DNA damage repair can affect the production of new tumor antigens and alter the immune microenvironment, thereby affecting tumor immunotherapy(Abou Khouzam et al. 2020). Delayed or incorrect repair of DNA damage can lead to alterations in the tumor genome, thereby changing the immune homeostasis in the tumor microenvironment(Luo et al. 2021). The interaction between glioma and the host immune system is being studied, and therapeutic attempts to activate the host immune system to kill tumor cells have shown some clinical efficacy(Li et al. 2021b). Despite advances in therapeutic approaches for lower-grade gliomas, the prognosis of LGG remains poor, even after surgical resection, due to the susceptibility to recurrence or transformation to HGGs. There is regular inactivation of certain DDR pathways during LGG onset and advancement, whereas mutations between DDR genes are also associated with the chemoresistance of tumor cells(Rocha et al. 2018). Therefore, DDR genes are associated with prognosis and could be utilized in predicting treatment response as well as overall prognosis for cancer patients.
LGG heterogeneity often favors limited therapeutic options and is also difficult to monitor for survival. Therefore, the identification of molecular phenotypic subtypes for LGG is of utmost importance. However, the role of DDR in LGG is still required to be revealed in a thorough manner. For characterizing the variations across DDR-based subtypes in LGG, we investigated multi-omics data from genomics, transcriptomics, and proteomics in this study. The differences in immunotherapeutic response and immune profiles among DDR-based subtypes have been explored by further studies. Our study showed that patients with lower-grade gliomas possess two different DDR states, namely, DDR-activated subtypes and DDR-suppressed subtypes. In the DDR-activated subgroup, patients exhibit aggressive clinical behaviour and have poor prognoses. To describe a molecular profile for the different DDR subtypes among LGG, there were considerable genomic alterations for two subtypes. IDH1 has been found to undergo mutation more often in the DDR suppressor subtype, while mutations in TP53 were not significantly different between the two groups. Mutations in the IDH1 gene identified glioma subtypes with various biological, clinical, and radiological features(Hartmann et al. 2009; Patel et al. 2017; Yan et al. 2009). LGG develops through early mutations in IDH1, leading to the accumulation of 2-hydroxyglutaric acid and genome-wide DNA damage repair, followed by the acquisition of two sets of co-occurring genetic alterations: mutations in TP53 and ATRX, or 1p/19q symbiosis and mutations in TERT, CIC, and FUBP1(Bettegowda et al. 2011; Jiao et al. 2012; Killela et al. 2014). However, determining whether the genetic alterations that drive the development and progression of IDH mutant gliomas in terms of whether they are derived from DNA damage repair is worth investigating. Due to the challenges encountered in directly targeting alterations driving IDH mutant gliomagenesis, future studies could focus on selectively targeting IDH mutant gliomas for immunotherapy and synthetic lethality in the DDR pathway.
Interestingly, among the DDR-suppressed subgroup, CIC is the second while EGFR is the third most frequently mutated in comparison to the DDR-activated subgroup. The protein encoded by CIC is a homolog of the Drosophila melanogaster tube Capicua gene, which is a member of the high mobility group (HMG) box superfamily of transcriptional repressors. This protein consists of a conserved HMG structural domain for DNA binding, nuclear localization, and a conserved C-terminal(Bettegowda et al. 2011). Some reports suggest CIC can widely activate gene expression through the independent EGFR pathway. CIC loss enhances tumor formation and reduces the latency of tumor development(Yang et al. 2017). This study identifies an essential role of CIC in the regulation of neuronal cell proliferation and spectrum specification. It also shows that CIC mutations influence the pathogenesis of oligodendrogliomas and strategies for associated targeted therapies.
We have also explored the role of DDR in the LGG immune microenvironment, where different immunological profiles exist for DDR subtypes. Activated (CD4 T cells, B cells, CD8 T cells, and DC cells), and natural killer cells were observed considerably upregulated in the DDR activation subgroup. Researchers demonstrated that KML001 (a telomere-targeting drug) may inhibit cell proliferation and cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machinery. As a result of treatment with KML001, dysfunctional telomere-induced foci (TIF), DNA damage marker H2AX, and topoisomerase cleavage complex (TOPcc) accumulation increased, leading to the attrition of telomeres(Cao et al. 2019). According to Zhao J et al., during HCV infection, inadequate ATM (DNA repair enzyme) results in increased damage to DNA. Consequently, HCV T cells are rendered prone to apoptosis which also contributes to the loss or dysregulation of naive T cells(Zhao et al. 2018). Moreover, selective induction of DNA repair pathways in human B cells activated by CD4 + T cells is also reported(Wu et al. 2010). The study by Galgano Alessia et al. shows that CD8 T cells engage unique DDRs during exponential division that is not observed in other exponentially dividing cells, in T lymphocytes after UV or X irradiation, or in non-metastatic tumor cells. When CD8 T cells divide, all DDR pathways as well as cell cycle checkpoints got affected, whereas only one DDR pathway is affected for other cell types(Galgano et al. 2015). These results illustrate the role of CD8 T cells in maintaining genome integrity despite their extensive division which highlights DDR’s fundamental role in the efficiency of CD8 immune responses. Karo Jenny M et al. reported that NK cells incapable of expressing either RAGs or RAG endonuclease activity during ontogeny display cell-intrinsic hyperresponsiveness but are less capable of surviving after virus-driven proliferation, reduced DNA damage response mediators expression, and defects in repairing DNA breaks(Karo et al. 2014). These findings suggest that DDR subtypes possess unique differences in terms of the infiltration of immune cells, implying varied immunotherapeutic responses among subtypes.
We adjusted mRNA levels according to DDR subtypes, classified them into high and low expression groups based on median mRNA expression, and constructed DDR Score. We explored the gene expression analysis from the IMvigor210 cohort for response-related genes and found that patients with high DDR score scores in the CR (complete response)/ PR (Partial response) group were fewer, and the CR/PR group had higher DDR signature scores compared to the SD (stable disease)/PD (progressive disease) group. We combined the six DDR genomes as a DDR subtype identification signature that showed excellent performance in training and validating cohorts to classify patients into different DDR subtypes. Further, we were able to identify 11 drugs possessing considerable sensitivity in the prognostic model (A-443654, A-770041, Acadesine, Benzamide, Motesanib diphosphate, Navitoclax, Ponatinib, Rucaparib phosphate Saracatinib, Tretinoin, Veliparib dihydrochloride), which may enhance LGG's clinical effectiveness.
Numerous studies have identified that DDR hub genes (TRIP13, CDK1, CDK2, TYMS, SMC4, and WEE1) play an essential role in tumor progression and metastasis. Thyroid hormone receptor-interacting factor 13 (TRIP13) is an important regulator of spindle assembly checkpoint and double-strand break repair. As per previous reports, TRIP13 is identified as aberrantly expressed among several human cancers, whereas TRIP13 knockdown inhibits cell proliferation, induces cell cycle arrest and promotes apoptosis, impairs cell viability, and finally interferes with the growth of tumor xenografts. In addition, TRIP13 can directly bind to the epithelial growth factor receptor (EGFR) and regulate the EGFR signaling pathway(Gao et al. 2019). Recent studies have indicated that high expression of TRIP13 promoted the proliferation and migration of ESCC cells and induced NDP resistance via enhancing the repair of DNA damage and inhibiting apoptosis(Zhang et al. 2022). Additionally, TRIP13 overexpression restored the impacts of miR-129-5p overexpression on malignant cell phenotypes and cell cycle. MiR-129-5p down-regulated TRIP13 expression, thereby restraining the malignant progression of CRC cells(Cao et al. 2022). Cycle protein-dependent kinase (CDK) is an essential member of the protein kinase family and among them, CDK1 and CDK2 play critical roles in cell cycle regulation, checkpoint activation, and DNA damage repair(Li et al. 2021a). In human cells, there are mainly the following CDKs that regulate the cell cycle by binding to the corresponding cyclin: 1) CDK2 promotes G1 / s transition by binding to cyclinE and promotes replication initiation and S phase by binding to cyclinA; 2) CDK1 interacts with cyclinB, whose expression in G2 / M phase increases gradually and reaches the peak, to activate cells to enter M phase, maintain M phase, ensure normal mitosis, and prevent cells from entering G1 phase ahead of time(Liu et al. 2020; Tadesse et al. 2020). In vitro studies have shown that CDK2 is responsible for promoting the G1/S transition and DNA replication initiation in normal cells. When CDK2 is absent, CDK1 can completely compensate for the function of CDK2 by promoting cell entry into S-phase through binding to cyclinE and DNA replication initiation through binding to cyclinA(Roskoski 2019). In addition, CDK1 can fully compensate for the absence of CDK2 in cell cycle regulation and has many other vital functions that cannot be replaced by CDK2, such as CDK1 capacity of promoting DNA double-strand break repair by homologous recombination and cell cycle checkpoint activation through phosphorylation of BRCA1(Kalra et al. 2017; Leal-Esteban and Fajas 2020). Thus, CDK1 and CDK2 are central to regulating many biological processes, including cell cycle regulation, DNA replication, and DNA damage repair, and closely link these biological processes to the cell cycle process. Further, the downstream target of FOXM1 is TYMS. TYMS knockdown reversed the 5-FU resistance caused by FOXM1 overexpression and re-sensitized HCC cells to 5-FU treatment. This reveals that TYMS functions as an oncogene in HCC and that inhibiting the FOXM1-TYMS axis may increase patient survival and offer novel treatment options for individuals with advanced HCC.(Wang et al. 2022). Li et al., proved that SMC4 which has the highest positive regression coefficient in glioma risk model is strongly linked with malignant progression and TMZ resistance of gliomas in an E2F1-dependent manner(Li et al. 2022). Lal Shruti et al. demonstrated that WEE1 inhibition in pancreatic cancer cells is dependent on DNA repair status in a context-dependent manner(Lal et al. 2016). Finally, we applied qRT-PCR for measuring the expression levels of six DDR genes. The results showed that our samples shared the same expression pattern as the ones from the public database.
Our study is not without limitations. First, further studies with more different expression detection platforms are required for the purpose of validating the optimum cut-off values in identifying the DDR subtype. Second, we focused only on multiple cohort data to provide reliable DDR-related survival information and molecular characterization. On the other hand, future exploration of in vivo and in vitro mechanisms could provide additional information on DDR subtype alterations. In the context of this, our follow-up research will aim to confirm the findings of this study in terms of clinical relevance as well as molecular mechanisms.